PT  - JOURNAL ARTICLE
AU  - Johanna Tomorsky
AU  - Philip R. L. Parker
AU  - Chris Q. Doe
AU  - Cristopher M. Niell
TI  - Precise levels of Nectin-3 and an interaction with Afadin are required for proper synapse formation in postnatal visual cortex
AID  - 10.1101/870691
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 870691
4099  - http://biorxiv.org/content/early/2020/06/13/870691.short
4100  - http://biorxiv.org/content/early/2020/06/13/870691.full
AB  - Background Developing cortical neurons express a tightly choreographed sequence of cytoskeletal and transmembrane proteins to form and strengthen specific synaptic connections during circuit formation. Nectin-3 is a cell-adhesion molecule with previously described roles in synapse formation and maintenance. This protein and its binding partner, Nectin-1, are selectively expressed in upper-layer neurons of mouse visual cortex, but their role in the development of cortical circuits is unknown.Methods Here we block Nectin-3 expression (via shRNA) or overexpress Nectin-3 in developing layer 2/3 visual cortical neurons using in utero electroporation. We then assay dendritic spine densities at three developmental time points: eye opening (postnatal day (P)14), one week following eye opening after a period of heightened synaptogenesis (P21), and at the close of the critical period for ocular dominance plasticity (P35).Results Knockdown of Nectin-3 beginning at E15.5 or ∼P19 increased dendritic spine densities at P21 or P35, respectively. Conversely, overexpressing full length Nectin-3 at E15.5 led to decreased dendritic spine densities when all ages were considered together. Interestingly, an even greater decrease in dendritic spine densities, particularly at P21, was observed when we overexpressed Nectin-3 lacking its Afadin binding domain, indicating Afadin may facilitate spine morphogenesis after eye opening.Conclusion These data collectively suggest that the proper levels of Nectin-3, as well as the interaction of Nectin-3 with Afadin, facilitate normal synapse formation after eye opening in layer 2/3 visual cortical neurons.Competing Interest StatementThe authors have declared no competing interest.PAJPuncta Adherentia JunctionNec3-shRNANectin-3 short hairpin RNANec3-OENectin-3 overexpressionNec3ΔafadinNectin-3 missing the Afadin binding sitesiRNAsmall interfering RNAV1Primary visual cortexL2/3Layer 2/3P14Postnatal day 14ODPOcular dominance plasticityIHCimmunohistochemistry.